NASBA Coupled to Paper Microfluidics for RNA Detection.

The analysis of RNA sequences is crucial to obtain invaluable insights into disease prognosis. Reliable and rapid diagnostic solutions at the site of sample collection contribute toward optimal delivery of medical treatment. For this reason, the development of more sensitive and portable RNA detection techniques are expected to advance current point-of-care (POC) diagnostic capabilities. Advancements of POC diagnostic technologies will also contribute to counter the spread of emerging viruses. Reverse transcriptase polymerase chain reaction (RT-PCR) is the most commonly used technique to identify etiological organisms of infections. However, the need for thermocycler and fluorescent measurement renders RT-PCR less suitable for POC applications. Here, we provide a step-by-step protocol of Nucleic Acid Sequence-Based Amplification (NASBA), a robust isothermal RNA amplification technique, coupled with a portable paper microfluidics detection format.

A Single Electrochemical Biosensor Designed to Detect Any Virus.

Viruses are the primary cause of many infectious diseases in both humans and animals. Various testing methods require an amplification step of the viral RNA sample before detection, with quantitative reverse transcription polymerase chain reaction (RT-qPCR) being one of the most widely used along with lesser-known methods like Nucleic Acid Sequence-Based Amplification (NASBA). NASBA offers several advantages, such as isothermal amplification and high selectivity for specific sequences, making it an attractive option for low-income facilities. In this research, we employed a single electrochemical biosensor (E-Biosensor) designed for potentially detecting any virus by modifying the NASBA protocol. In this modified protocol, a reverse primer is designed with an additional 22-nucleotide sequence (tag region) at the 5'-end, which is added to the NASBA process. This tag region becomes part of the final amplicon generated by NASBA. It can hybridize with a single specific E-Biosensor probe set, enabling subsequent virus detection. Using this approach, we successfully detected three different viruses with a single E-Biosensor design, demonstrating the platform's potential for virus detection.

Detection of Multiplex NASBA RNA Products Using Colorimetric Split G Quadruplex Probes.

Structural RNA is a challenging target for recognition by hybridization probes. This chapter addresses the recognition problem of RNA amplicons in samples obtained by multiplex nucleic acid sequence-based amplification (NASBA). The method describes the design of G-quadruplex binary (split) DNA peroxidase sensors that produces colorimetric signal upon recognition of NASBA amplicons.

Rapid and effective detection of Macrobrachium rosenbergii nodavirus using a combination of nucleic acid sequence-based amplification test and immunochromatographic strip.

Nucleic acid sequence-based amplification (NASBA) provides a fast and convenient approach for nucleic acid amplification under isothermal conditions, and its combination with an immunoassay-based lateral flow dipstick (LFD) could produce a higher detection efficiency for M. rosenbergii nodavirus isolated from China (MrNV-chin). In this study, two specific primers and a labelled probe of the capsid protein gene of MrNV-chin were constructed. The process of this assay mainly included a single-step amplification at a temperature of 41 â„ƒ for 90 min, and hybridization with an FITC-labeled probe for 5 min, with the hybridization been required for visual identification during LFD assay. The test results indicated that, the NASBA-LFD assay showed sensitivity for 1.0 fg M. rosenbergii total RNA with MrNV-chin infection, which was 104 times that of the present RT-PCR approach for the detection of MrNV. In addition, no products were created for shrimps with infection of other kinds of either DNA or RNA virus, which indicated that the NASBA-LFD was specific for MrNV. Therefore, the combination of NASBA and LFD is a new alternative detection method for MrNV which is rapid, accurate, sensitive and specific without expensive equipment and specialised personnel. Early detection of this infectious disease among aquatic organisms will help implement efficient therapeutic strategy to prevent its spread, enhance animal health and limit loss of aquatic breeds in the event of an outbreak.

Evaluation of real-time NASBA assay for the detection of SARS-CoV-2 compared with real-time PCR.

PURPOSE: In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2. METHODS: Primers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed. RESULTS: The limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively. CONCLUSIONS: The results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.

A rapid and high sensitivity RNA detection based on NASBA and G4-ThT fluorescent biosensor.

In recent years, various newly emerged and re-emerged RNA viruses have seriously threatened the global public health. There is a pressing need for rapid and reliable nucleic acid-based assays for detecting viral RNA. Here, we successfully developed a highly sensitive, easy-to-operate G4-ThT-NASBA system to detect viral RNA that no need for labeled primers and probes. Next, we tested the system for detecting the Classical Swine Fever Virus (CSFV), an RNA virus that causes a highly contagious disease in domestic pigs and wild boar and easily causes huge economic losses. Results showed that the system, integrated the G4-ThT fluorescent biosensor and NASBA (Nuclear acid sequence-based amplification),is capable to detect as little as 2 copies/µL of viral RNA without interfering by other swine viral RNA. Moreover, we were able to detect CSFV RNA within 2 h in serum samples taken from the field in a real-time mode. These findings indicate that the G4-ThT-NASBA system is a rapid, high sensitivity and easy-to-operate technique for RNA detection. The method also has the real-time detection capability which may be easily integrated in a highly automated system such as microfluidic chips.

An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein.

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

Detection of micro-RNA by a combination of nucleic acid sequence-based amplification and a novel chemiluminescent pyrophosphate assay.

Micro-RNA has attracted much attention as a biomarker for disease progression and malignancy. A compact, simple, rapid, and highly sensitive method is required to perform simple genetic analyses, such as point-of-care testing (POCT), at the clinic or bedside. Nucleic acid sequence-based amplification (NASBA) is a specific amplification method for a single-stranded RNA fragment that is useful for the highly sensitive detection of miRNAs. In this work, we developed a novel miRNA analytical system for POCT by combining the NASBA and chemiluminescence methods. Because the NASBA reaction is conducted at a constant temperature (41°C) and detection by chemiluminescence reaction does not require a light source, these methods could be combined to amplify 100 ng/assay miRNA. This combined miRNA detection method could be useful for the future development of compact POCT systems.

A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin.

Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

Application and evaluation of nucleic acid sequence-based amplification, PCR and cryptococcal antigen test for diagnosis of cryptococcosis.

BACKGROUND: Cryptococcosis is a major opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. In the early stages of cryptococcosis, limitations of the detection method may hinder the diagnosis. A molecular diagnostic technique based on nucleic acid sequence-based amplification (NASBA) method was developed to fulfil the need for efficient diagnosis of cryptococcosis. METHODS: We compared the diagnostic performance of NASBA, PCR and cryptococcal antigen (CrAg) test (colloidal gold method) in clinical samples from 25 cryptococcosis patients (including 8 cryptococcal meningoencephalitis and 17 pulmonary cryptococcosis) who were categorized as proven cases (n = 10) and probable cases (n = 15) according to the revised EORTC/MSG definitions. 10 patients with non-Cryptococcus infection and 30 healthy individuals were categorized as control group. RESULTS: The lowest detection limit of NASBA was 10 CFU/mL, and RNA of non-target bacteria or fungi was not amplified. The sensitivity of NASBA, PCR and colloidal gold method was 92.00% (95% CI 72.50-98.60%), 64.00% (95% CI 42.62-81.29%), 100.00% (95% CI 83.42-100.00%), and the specificity was 95.00% (95% CI 81.79-99.13%), 80.00% (95% CI 63.86-90.39%) and 82.50% (95% CI 66.64-92.11%) respectively. The highest specificity (97.50%), accuracy (95.38%) and k value (0.90) were achieved when both NASBA and colloidal gold results were positive. CONCLUSIONS: NASBA is a new alternative detection method for cryptococcosis which is both accurate and rapid without expensive equipment and specialised personnel. It may be used as a tool for confirming current infection as well as monitoring the effectiveness of antifungal treatment. The use of NASBA to detect Cryptococcus RNA in blood samples is of great significance for the diagnosis of pulmonary cryptococcosis. The combination of NASBA and colloidal gold can improve the diagnostic accuracy of cryptococcosis.

Ultrasensitive version of nucleic acid sequence-based amplification (NASBA) utilizing a nicking and extension chain reaction system.

Nucleic acid sequence-based amplification (NASBA) is a transcription-based isothermal amplification technique especially designed for the detection of RNA targets. The NASBA basically relies on the linear production of T7 RNA promoter-containing double-stranded DNA (T7DNA), and thus the final amplification efficiency is not sufficiently high enough to achieve ultrasensitive detection. We herein ingeniously integrate a nicking and extension chain reaction system into the NASBA to establish an ultrasensitive version of NASBA, termed Nicking and Extension chain reaction System-Based Amplification (NESBA). By employing a NESBA primer set designed to contain an additional nicking site at the 5' end of a NASBA primer set, the T7DNA is exponentially amplified through continuously repeated nicking and extension chain reaction by the combined activities of nicking endonuclease (NE) and reverse transcriptase (RT). As a consequence, a much larger number of RNA amplicons would be produced through the transcription of the amplified T7DNA, greatly enhancing the final fluorescence signal from the molecular beacon (MB) probe binding to the RNA amplicon. Based on this unique design principle, we successfully identified the target respiratory syncytial virus A (RSV A) genomic RNA (gRNA) down to 1 aM under isothermal conditions, which is 100-fold more sensitive than regular NASBA.

The mechanism and improvements to the isothermal amplification of nucleic acids, at a glance.

A comparative review of the most common isothermal methods is provided. In the last two decades, the challenge of using isothermal amplification systems as an alternate to the most extensive and long-standing nucleic acids-amplifying method-the polymerase chain reaction-has arisen. The main advantage of isothermal amplification is no requirement for expensive laboratory equipment for thermal cycling. Considerable efforts have been made to improve the current techniques of nucleic acid amplification and the development of new approaches based on the main drawbacks of each method. The most important and challenging goal was to achieve a low-cost, straightforward system that is rapid, specific, accurate, and sensitive.

Rapid and simultaneous detection of Japanese encephalitis virus by real-time nucleic acid sequence-based amplification.

Japaneses encephalitis (JE) is most common zoonoses caused by Japanese encephalitis virus (JEV) with a high mortality and disability rate. To take timely preventive and control measures, early and rapid detection of JE RNA is necessary. But due to characteristic brief and low viraemia, JE RNA detection remains challenging. In this study, a real-time nucleic acid sequence-based amplification (RT-NASBA) was developed for rapid and simultaneous detection of JEV. Four pairs of primer were designed using a multiple genome alignment of all JEV strains from GenBank. NASBA assay established and optimal reaction conditions were confirmed by using primers and probe on ns1 gene of JEV. The specificity and sensitivity of the assay were compared with RT-PCR by using serial RNA and virus cultivation dilutions. The results showed that JEV RT-NASBA assay was established, and robust signals could be observed in 10 min with high specificity. The limit of dectetion of RT-NASBA was 6 copies per reaction. The assay was thus 100 to 1, 000 times more sensitive than RT-PCR. The cross-reaction was performed with other porcine pathogens, and negative amplification results indicated the high specificity of this method. The novel JEV RT-NASBA assay could be used as an efficient molecular biology tool to diagnose JEV, which would facilitate the surveillance of reproductive failure disease in swine and would be beneficial for public health security.

A highly sensitive and rapid enzymatic method using a biochemical automated analyzer to detect inorganic pyrophosphate generated by nucleic acid sequence-based amplification.

BACKGROUND AND AIMS: Polymerase chain reaction-based techniques require expensive equipment for fluorescence detection of the products. However, the measurement of inorganic pyrophosphate (PPi) released during DNA synthesis can be used to quantify target genes without such equipment. Here, we devised a high-sensitivity enzymatic assay for detection of PPi. MATERIALS AND METHODS: In our assay method, PPi was converted to hypoxanthine by hypoxanthine phosphoribosyl transferase. Xanthine dehydrogenase converted the hypoxanthine to uric acid and yielded two molecules of NADH, which in turn reduced Fe3+ to Fe2+ (mediated by 1-methoxy-5-ethylphenazinium ethylsulfate). 2-Nitroso-5-(N-propyl-N-sulfopropylamino) phenol chelated the Fe2+, which resulted in an intensely colored product that could be measured using a biochemical automated analyzer. RESULTS: The assay was able to detect PPi within 10 min. It was linear between 0 and 10 µmol/L PPi, and intra-run and inter-run coefficients of variation were 1%-2%. Other validation tests with a biochemical automated analyzer were satisfactory. The assay could potentially be used to directly quantify samples after isothermal nucleic acid sequence-based amplification of a target gene. CONCLUSION: The method developed here for detection of PPi can be used to measure nucleic acid biomarkers in biological samples in clinical practice using a high-throughput biochemical automated analyzer.

Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 infection, past or present, in patients. We also discuss ongoing research on isothermal techniques, CRISPR-mediated detection assays, and point-of-care diagnostics that have potential for use in SARS-CoV-2 detection. Large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. The low-cost isothermal technologies described in this Review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks.

Point-of-care tests for malaria: speeding up the diagnostics at the bedside and challenges in malaria cases detection.

Malaria remains as one of the major public health problems worldwide. About 228 million cases occurred in 2018 only, with Africa bearing about 93% of the cases. Asymptomatic population carrying the various forms of the parasite Plasmodium in endemic areas plays an important role in the spread of the disease. To tackle this battle, more sensitive and precise detection kits for malaria are crucial to better control the number of new malaria cases. In this review, we not only discuss some of the available approaches to rapidly detect new malaria cases in endemic areas but also shed light on parallel problems that may affect the detection of individuals infected with the parasite, covering kelch 13 mutation, glucose 6-phosphate dehydrogenase deficiency, and hemoglobin disorders. Available approaches for malaria detection covered in this review are focused on point-of-care tests, including portable polymerase chain reaction and aptamers.

Isothermal nucleic acid amplification techniques for detection and identification of pathogenic fungi: A review.

BACKGROUND: Fungal infections have increased during the last years due to the AIDS epidemic and immunosuppressive therapies. The available diagnostic methods, such as culture, histopathology and serology, have several drawbacks regarding sensitivity, specificity and time-consuming, while molecular methods are still expensive and dependent on many devices. In order to overcome these challenges, isothermal nucleic acid amplification techniques (INAT) arose as promising diagnostic methods for infectious diseases. OBJECTIVE: This review aimed to present and discuss the main contributions of the isothermal nucleic acid amplification techniques applied in medical mycology. METHODS: Papers containing terms for each INAT (NASBA, RCA, LAMP, CPA, SDA, HAD or PSR) and the terms 'mycoses' or 'disease, fungal' were obtained from National Center for Biotechnology Information database until August 2019. RESULTS: NASBA, RCA, LAMP and PSR are the INAT reported in the literature for detection and identification of pathogenic fungi. Despite the need of a previous conventional PCR, the RCA technique might also be used for genotyping or cryptic species differentiation, which may be important for the treatment of certain mycoses; nevertheless, LAMP is the most used INAT for pathogen detection. CONCLUSION: Among all INATs herein reviewed, LAMP seems to be the most appropriate method for fungal detection, since it is affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free and deliverable to end-users, fulfilling all ASSURED criteria of the World Health Organization for an ideal diagnostic method.

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.

[Application of NASBA and RPA in detection of pathogenic bacteria].

Nucleic acid sequence-based amplification and recombinase polymerase amplification are the recently developed thermostatic amplification techniques based on PCR. This paper briefly summarizes the principle of reaction, design principle of primer and probe, advantage of these two techniques (simple, accurate, highly sensitive and rapid) and introduces the application of the techniques in the detection of pathogenic bacteria.

Pooled analysis of nuclear acid sequence-based amplification for rapid diagnosis of Mycoplasma pneumoniae infection.

BACKGROUND: Mycoplasma pneumoniae (M pneumoniae) is a common human etiology of respiratory infections. Nuclear acid sequence-based amplification (NASBA) shows good value for the detection of M pneumoniae that surpasses PCR. However, the optimal detection technology still remains to be identified. The purpose of this meta-analysis was to systematically evaluate the overall accuracy of NASBA for diagnosing M pneumoniae infections. METHODS: The databases PubMed, Cochrane Library, Google Scholar, CNKI, Wang Fang, and Baidu Scholar were comprehensively searched from their initiation date to December 2017 for NASBA in the diagnosis of M pneumoniae infection. Meta-DiSc 1.4 statistical software was used to evaluate the sensitivity (SEN), specificity (SPE), negative likelihood ratio (-LR), positive likelihood ratio (+LR), diagnostic odds ratio (DOR), and summary receiver operating characteristic (SROC). RevMan 5.2 statistical software was used for quality evaluation of the included articles. Publication bias was evaluated by funnel plot. RESULTS: Six articles with high quality, including 10 studies, were finally included in this meta-analysis. The combined statistics results for the diagnosis of M pneumoniae infection by NASBA were 0.77 (SEN, 95% CI: 0.71 to 0.82); 0.98 (SPE, 95% CI: 0.98 to 0.99); 0.22 (-LR, 95% CI: 0.13 to 0.39); 50.38 (+ LR, 95% CI: 21.85 to 116.17); 292.72 (DOR, 95% CI: 95.02 to 901.75); and 0.9875 (the area under the curve of SROC). CONCLUSION: Nuclear acid sequence-based amplification is a reliable technique to diagnose M pneumoniae infection. However, whether it can replace PCR and serology need to be further studied.